Method for promoting muscle regeneration by administering an antibody to the IL-6 receptor

ABSTRACT

The present inventors studied the effects of inhibiting the IL-6 signaling pathway on muscle cell growth. As a result, they discovered that, administering an IL-6 inhibitor can promote the adhesion, proliferation, and differentiation of satellite cells and therefore muscle regeneration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International ApplicationSerial No. PCT/JP2007/057745, filed on Apr. 6, 2007, which claims thebenefit of Japanese Application Serial No. 2006-106445, filed on Apr. 7,2006. The contents of the foregoing applications are hereby incorporatedby reference in their entirety.

TECHNICAL FIELD

The present invention relates to agents for promoting muscleregeneration, which comprise an IL-6 inhibitor as an active ingredient,and uses thereof.

BACKGROUND ART

Muscle atrophy is known to occur in paravertebral muscles, lower-limbsoleus muscles, and such following exposure to a space environment whichis a microgravity environment, a long-term bedrest, or a plastercast-immobilized state. Damage and necrosis of skeletal muscles arecompensated by regeneration, and when muscle regeneration does not fullycompensate for the necrosis of muscle fibers, muscle atrophy is thoughtto occur. It is known that in the regeneration process followingskeletal muscle damage, satellite cells are recruited. Satellite cellsare tissue-specific stem cells normally existing as a quiescent(inactive) state in skeletal muscles. They proliferate anddifferentiate, and fuse with muscle fibers to promote muscleregeneration. However, the factors that promote satellite cellrecruitment, growth, and differentiation in vivo have not been clarifiedyet.

IL-6 is a cytokine called B-cell stimulating factor 2 (BSF2) orinterferon β2. IL-6 was discovered as a differentiation factor involvedin the activation of B-cell lymphocytes (Non-patent Document 1), and waslater revealed to be a multifunctional cytokine that influences thefunction of various cells (Non-patent Document 2). IL-6 has beenreported to induce maturation of T lymphocyte cells (Non-patent Document3).

IL-6 transmits its biological activity via two kinds of proteins on thecell. One of the proteins is the IL-6 receptor which is a ligand bindingprotein to which IL-6 binds and has a molecular weight of about 80 kDa(Non-patent Document 4; and Non-patent Document 5). In addition to amembrane-bound form that penetrates and is expressed on the cellmembrane, the IL-6 receptor is also present as a soluble IL-6 receptorwhich mainly consists of the extracellular region of the membrane-boundform.

The other is the membrane protein gp130 which has a molecular weight ofabout 130 kDa and is involved in non-ligand binding signal transduction.The biological activity of IL-6 is transmitted into the cell throughformation of the IL-6/IL-6 receptor complex by IL-6 and IL-6 receptorand binding of the complex with gp130 thereafter (Non-patent Document6).

IL-6 inhibitors are substances that inhibit the transmission of IL-6biological activity. Until now, antibodies against IL-6 (anti-IL-6antibodies), antibodies against IL-6 receptors (anti-IL-6 receptorantibodies), antibodies against gp130 (anti-gp130 antibodies), IL-6variants, partial peptides of IL-6 or IL-6 receptors, and such have beenknown.

There are several reports regarding the anti-IL-6 receptor antibodies(Non-patent Document 7; Non-patent Document 8; Patent Document 1; PatentDocument 2; and Patent Document 3). A humanized PM-1 antibody, which hadbeen obtained by transplanting into a human antibody, thecomplementarity determining region (CDR) of mouse antibody PM-1(Non-patent Document 9), which is one of anti-IL-6 receptor antibodies,is known (Patent Document 4).

To date, insulin-like growth factor-I (Non-patent Document 10) andanti-myostatin antibodies (Non-patent Document 11) have been known tosuppress muscle atrophy and promote muscle regeneration. However, it isnot clear whether cytokines, such as IL-6, influence muscle regenerationor not.

Documents of related prior arts for the present invention are describedbelow.

-   [Patent Document 1] International Patent Application Publication No.    WO 95/09873.-   [Patent Document 2] French Patent Application No. FR 2694767.-   [Patent Document 3] U.S. Pat. No. 5,216,128.-   [Patent Document 4] WO 92/19759.-   [Non-patent Document 1] Hirano, T. et al., Nature (1986) 324, 73-76.-   [Non-patent Document 2] Akira, S. et al., Adv. in Immunology (1993)    54, 1-78.-   [Non-patent Document 3] Lotz, M. et al., J. Exp. Med. (1988) 167,    1253-1258.-   [Non-patent Document 4] Taga, T. et al., J. Exp. Med. (1987) 166,    967-981.-   [Non-patent Document 5] Yamasaki, K. et al., Science (1988) 241,    825-828.-   [Non-patent Document 6] Taga, T. et al., Cell (1989) 58, 573-581.-   [Non-patent Document 7] Novick, D. et al., Hybridoma (1991) 10,    137-146.-   [Non-patent Document 8] Huang, Y. W. et al., Hybridoma (1993) 12,    621-630.-   [Non-patent Document 9] Hirata, Y et al., J. Immunol. (1989) 143,    2900-2906.-   [Non-patent Document 10] Barton-Davis, E. R. et al., Proc. Natl.    Acad. Sci. USA (1998) 95, 15603-15607.-   [Non-patent Document 11] Bogdanovich, S. et al, Nature (2002) 420,    418-421.-   [Non-patent Document 12] Dangott B. et al., Int J. Sports    Med. (2000) 21, 13-16.-   [Non-patent Document 13] Darr K C. and Schultz E., J. Appl.    Physiol. (1989) 67, 1827-1834.-   [Non-patent Document 14] Garry D J. et al., PNAS (2000) 97,    5416-5421.-   [Non-patent Document 15] Garry D J. et al., Dev. Biol. (1997) 188,    280-294.-   [Non-patent Document 16] Jejurikar S S. et al., Plast Reconstr    Surg (2002) 110, 160-168.-   [Non-patent Document 17] Mauro A., J. Biochem Cytol. (1961) 9,    493-498.-   [Non-patent Document 18] McCormick K M and Schultz E., Dev.    Dyn. (1994) 199, 52-63.-   [Non-patent Document 19] Moss F P. and Leblond C P., Anat.    Rec. (1971) 170, 421-435.-   [Non-patent Document 20] Mozdziak P E. et al., Biotech.    Histochem. (1994) 69, 249-252.-   [Non-patent Document 21] Mozdziak P E. et al., J. Appl.    Physiol. (2000) 88, 158-164.-   [Non-patent Document 22] Mozdziak P E. et al., J. Appl.    Physiol. (2001) 91, 183-190.-   [Non-patent Document 23] Mozdziak P E. et al., Eur. J. Appl.    Physiol. Occup. Physiol. (1998) 78, 136-40.-   [Non-patent Document 24] Schultz E., Dev. Biol. (1996) 175, 84-94.-   [Non-patent Document 25] Schultz E. et al., J. Appl. Physiol. (1994)    76, 266-270.-   [Non-patent Document 26] Schultz E. et al., Muscle Nerve. (1985) 8,    217-222.-   [Non-patent Document 27] Snow M H., Anat. Rec. (1977) 188, 181-199.-   [Non-patent Document 28] Snow M H., Anat. Rec. (1990) 227, 437-446.-   [Non-patent Document 29] Wang X D., Am. J. Physiol. Cell    Physiol. (2006) 290, C981-C989.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention was achieved in view of the above circumstances.One of the objectives in the present invention is to provide agents forpromoting muscle regeneration, which comprise an IL-6 inhibitor as anactive ingredient.

Another objective of the present invention is to provide methods forpromoting muscle regeneration, which comprise the step of administeringan IL-6 inhibitor to subjects with muscle atrophy.

Means for Solving the Problems

To solve the problems described above, the present inventors studied theeffects of inhibiting the IL-6 signaling pathway on muscle cell growth.

First, C2C12 cells were cultured in a differentiation medium containingvarious concentrations of MR16-1 (an anti-mouse IL-6 receptor monoclonalantibody), and proteins involved in muscle regeneration (MyoD, myogenin,myogenic regulatory factor proteins, and myosin heavy chain) weredetected by immunohistochemical analyses. Furthermore, the expression ofM-cadherin, phospho-p38, and MyoD, which are muscle differentiationmarkers, was confirmed by Western blot analysis.

The result revealed that C2C12 cell proliferation was suppressed by theaddition of MR16-1; however, the percentage distribution of C2C12 cellsexpressing MyoD, myogenin, myogenic regulatory factor proteins, andmyosin heavy chain increases. Furthermore, the expression levels ofM-cadherin, phospho-p38, and MyoD increased in cells treated withMR16-1. These results indicated that the immune system plays animportant role in the development and/or growth of muscle fibers throughthe IL-6 signaling pathway.

Next, the present inventors used male mice (C57BL/6J Jcl) to examine thechanges of the reactions of satellite cells in response to MR16-1supplementation to the loaded or unloaded whole single soleus musclefibers.

As a result, MR16-1 treatment showed no specific effect on fiber atrophyor decrease of the number of satellite cells in response to unloading.However, the number of proliferation-activated satellite cells inresponse to reloading was revealed to increase following MR16-1treatment. Since satellite cells play an important role in regulatingthe mass of muscle fiber, it was suggested that the inhibition of IL-6might be a potential method for promoting muscle regeneration.

Specifically, the present inventors discovered that, administration ofan IL-6 inhibitor can promote the adhesion, proliferation, anddifferentiation of satellite cells and thus muscle regeneration ormuscle fiber enlargement are stimulated. Thereby, the present inventionwas completed.

More specifically, the present invention provides:

[1] an agent for promoting muscle regeneration, which comprises an IL-6inhibitor as an active ingredient;

[2] the agent of [1] for promoting muscle regeneration, wherein the IL-6inhibitor is an antibody that recognizes IL-6;

[3] the agent of [1] for promoting muscle regeneration, wherein the IL-6inhibitor is an antibody that recognizes an IL-6 receptor;

[4] the agent of [2] or [3] for promoting muscle regeneration, whereinthe antibody is a monoclonal antibody;

[5] the agent of [2] or [3] for promoting muscle regeneration, whereinthe antibody is an antibody that recognizes human IL-6 or a human IL-6receptor;

[6] the agent of [2] or [3] for promoting muscle regeneration, whereinthe antibody is a recombinant antibody;

[7] the agent of [6] for promoting muscle regeneration, wherein theantibody is a chimeric, humanized, or human antibody;

[8] the agent of any one of [1] to [7] for promoting muscleregeneration, wherein the muscle regeneration is muscle regenerationfrom muscle atrophy;

[9] a method for promoting muscle regeneration in a subject, whichcomprises the step of administering an IL-6 inhibitor to the subject;

[10] the method of [9], wherein the subject is affected with muscleatrophy;

[11] the method of [9] or [10], wherein the IL-6 inhibitor is anantibody that recognizes IL-6;

[12] the method of [9] or [10], wherein the IL-6 inhibitor is anantibody that recognizes IL-6 receptor;

[13] the method of [11] or [12], wherein the antibody is a monoclonalantibody;

[14] the method of [11] or [12], wherein the antibody is an antibodythat recognizes human IL-6 or human IL-6 receptor;

[15] the method of [11] or [12], wherein the antibody is a recombinantantibody;

[16] the method of [15], wherein the antibody is a chimeric, humanized,or human antibody;

[17] a use of an IL-6 inhibitor in producing an agent for promotingmuscle regeneration;

[18] the use of [17], wherein the IL-6 inhibitor is an antibody thatrecognizes IL-6;

[19] the use of [17], wherein the IL-6 inhibitor is an antibody thatrecognizes an IL-6 receptor;

[20] the use of [18] or [19], wherein the antibody is a monoclonalantibody;

[21] the use of [18] or [19], wherein the antibody is an antibody thatrecognizes human IL-6 or a human IL-6 receptor;

[22] the use of [18] or [19], wherein the antibody is a recombinantantibody; and

[23] the use of [22], wherein the antibody is a chimeric, humanized, orhuman antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of Western blot confirming protein expression inC2C12 cells. The effects of MR16-1 addition on the growth of C2C12 cellscultured for three days in a differentiation medium containing 2% horseserum were investigated.

FIGS. 2A-D are diagrams showing the effects of MR16-1 addition on theproperties of satellite cells in whole single fibers of soleus musclesof male mice (C57BL/6J Jcl) in the presence or absence of a load. FIG.2A shows the total number of BrdU-positive (mitotic active) satellitecells in muscle fibers sampled from tendon to tendon in each group.

In all the diagrams of FIG. 2 below, the symbols indicate groups in thefollowing states: Pre, group before hind-limb suspension; C, age-matchedcontrol group; CMR, age-matched control group treated with MR16-1; S,group with hind-limb suspension; SMR, group with hind-limb suspensiontreated with MR16-1. In all the graphs of FIG. 2 below, R+0 refers togroups immediately after seven days of housing or hind-limb suspension,and R+7 refers to groups seven days after reloading. In all the diagramsof FIG. 2 below, the data are presented as mean±SEM. * and †, P<0.05 vs.Pre and C in R+0 and S and SMR in R+0, respectively.

FIG. 2B is a diagram showing the total number of M-cadherin-positive(quiescent, resting) satellite cells in muscle fibers sampled fromtendon to tendon in each group.

FIG. 2C is a diagram showing the total number of satellite cells (bothBrdU-positive and M-cadherin-positive) in muscle fibers sampled fromtendon to tendon in each group.

FIG. 2D is a graph showing the percentage of BrdU-positive (mitoticactive) satellite cells/total satellite cells.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors discovered that muscle regeneration can bepromoted by supplementation of an anti-IL-6 receptor antibody. Thepresent invention is based on these findings.

The present invention relates to agents for promoting muscleregeneration, which comprise an IL-6 inhibitor as an active ingredient.

Herein, an “IL-6 inhibitor” is a substance that blocks IL-6-mediatedsignal transduction and inhibits IL-6 biological activity. Preferably,the IL-6 inhibitor is a substance that has inhibitory function againstthe binding of IL-6, IL-6 receptor, or gp130.

The IL-6 inhibitors of the present invention include, but are notlimited to, for example, anti-IL-6 antibodies, anti-IL-6 receptorantibodies, anti-gp130 antibodies, IL-6 variants, soluble IL-6 receptorvariants, and partial peptides of IL-6 or IL-6 receptors and lowmolecular weight compounds that show similar activities. Preferable IL-6inhibitors of the present invention include antibodies that recognizeIL-6 receptors.

The source of the antibody is not particularly restricted in the presentinvention; however, the antibody is preferably derived from mammals, andmore preferably derived from human.

The anti-IL-6 antibody used in the present invention can be obtained asa polyclonal or monoclonal antibody via known means. In particular,monoclonal antibodies derived from mammals are preferred as theanti-IL-6 antibody used in the present invention. The monoclonalantibodies derived from mammals include those produced from hybridomasand those produced from hosts transformed with an expression vector thatcomprises an antibody gene by genetic engineering methods. By binding toIL-6, the antibody inhibits IL-6 from binding to an IL-6 receptor andblocks the transmission of IL-6 biological activity into the cell.

Such antibodies include, MH166 (Matsuda, T. et al., Eur. J. Immunol.(1988) 18, 951-956), SK2 antibody (Sato, K. et al., transaction of the21^(st) Annual Meeting of the Japanese Society for Immunology (1991) 21,166), and so on.

Basically, anti-IL-6 antibody producing hybridomas can be prepared usingknown techniques as follows. Specifically, such hybridomas can beprepared by using IL-6 as a sensitizing antigen to carry outimmunization by a conventional immunization method, fusing the obtainedimmune cells with known parent cells by a conventional cell fusionmethod, and screening for monoclonal antibody-producing cells by aconventional screening method.

More specifically, anti-IL-6 antibodies can be produced as follows. Forexample, human IL-6 used as the sensitizing antigen for obtainingantibody can be obtained using the IL-6 gene and/or amino acid sequencesdisclosed in Eur. J. Biochem. (1987) 168, 543-550; J. Immunol. (1988)140, 1534-1541; and/or Agr. Biol. Chem. (1990) 54, 2685-2688.

After transforming an appropriate host cell with a known expressionvector system inserted with an IL-6 gene sequence, the desired IL-6protein is purified by a known method from the inside of the host cellor from the culture supernatant. This purified IL-6 protein may be usedas the sensitizing antigen. Alternatively, a fusion protein of the IL-6protein and another protein may be used as the sensitizing antigen.

Anti-IL6 receptor antibodies used for the present invention can beobtained as polyclonal or monoclonal antibodies by known methods. Inparticular, the anti-IL-6 receptor antibodies used in the presentinvention are preferably monoclonal antibodies derived from mammals. Themonoclonal antibodies derived from mammals include those produced fromhybridomas and those produced from hosts transformed with an expressionvector that comprises an antibody gene by genetic engineering methods.By binding to an IL-6 receptor, the antibody inhibits IL-6 from bindingto the IL-6 receptor and blocks the transmission of IL-6 biologicalactivity into the cell.

Such antibodies include, MR16-1 antibody (Tamura, T. et al., Proc. Natl.Acad. Sci. USA (1993) 90, 11924-11928); PM-1 antibody (Hirata, Y. etal., J. Immunol. (1989) 143, 2900-2906); AUK12-20 antibody, AUK64-7antibody and AUK146-15 antibody (WO 92/19759); and so on. Among them,the PM-1 antibody can be exemplified as a preferred monoclonal antibodyagainst the human IL-6 receptor, and the MR16-1 antibody as a preferredmonoclonal antibody against the mouse IL-6 receptor.

Basically, hybridomas producing an anti-IL-6 receptor monoclonalantibody can be prepared using known techniques as follows.Specifically, such hybridomas can be prepared by using an IL-6 receptoras the sensitizing antigen to carry out immunization by a conventionalimmunization method, fusing the obtained immune cells with a knownparent cell by a conventional cell fusion method, and screening formonoclonal antibody-producing cells by a conventional screening method.

More specifically, anti-IL-6 receptor antibodies can be produced asfollows. For example, a human IL-6 receptor or mouse IL-6 receptor usedas the sensitizing antigen for obtaining antibody can be obtained usingthe IL-6 receptor genes and/or amino acid sequences disclosed inEuropean Patent Application Publication No. EP 325474 and JapanesePatent Application Kokai Publication No. (JP-A) H03-155795,respectively.

There exist two kinds of IL-6 receptor proteins, i.e., protein expressedon the cell membrane and protein separated from the cell membrane(soluble IL-6 receptor) (Yasukawa, K. et al., J. Biochem. (1990) 108,673-676). The soluble IL-6 receptor consists essentially of theextracellular region of the cell membrane-bound IL-6 receptor, anddiffers from the membrane-bound IL-6 receptor in that it lacks thetransmembrane region or both the transmembrane and intracellularregions. Any IL-6 receptor may be employed as the IL-6 receptor proteinso long as it can be used as a sensitizing antigen for producing theanti-IL-6 receptor antibody utilized in the present invention.

After transforming an appropriate host cell with a known expressionvector system inserted with an IL-6 receptor gene sequence, the desiredIL-6 receptor protein is purified by a known method from the inside ofthe host cell or from the culture supernatant. This purified IL-6receptor protein may be used as a sensitizing antigen. Alternatively, acell expressing the IL-6 receptor or a fusion protein of the IL-6receptor protein and another protein may be used as a sensitizingantigen.

Anti-gp130 antibodies used in the present invention can be obtained aspolyclonal or monoclonal antibodies by known methods. In particular, theanti-gp130 antibodies used in the present invention are preferablymonoclonal antibodies derived from mammals. The monoclonal antibodiesderived from mammals include those produced from hybridomas and thoseproduced from hosts transformed with an expression vector that comprisesan antibody gene by genetic engineering methods. By binding to gp130,the antibody inhibits gp130 from binding to the IL-6/IL-6 receptorcomplex and blocks the transmission of IL-6 biological activity into thecell.

Such antibodies include, AM64 antibody (JP-A H03-219894); 4B11 antibodyand 2H4 antibody (U.S. Pat. No. 5,571,513); B-S12 antibody and B-P8antibody (JP-AH08-291199); and so on.

Basically, Anti-gp130 monoclonal antibody-producing hybridomas can beprepared using known techniques as follows. Specifically, suchhybridomas can be prepared by using gp130 as a sensitizing antigen tocarry out the immunization by a conventional immunization method, fusingthe obtained immune cells with a known parent cell by a conventionalcell fusion method, and screening for monoclonal antibody-producingcells by a conventional screening method.

More specifically, the monoclonal antibody can be produced as follows.For example, gp130 used as a sensitizing antigen for obtaining antibodycan be obtained using the gp130 gene and/or amino acid sequencedisclosed in European Patent Application Publication No. EP 411946.

After transforming an appropriate host cell with a known expressionvector system inserted with a gp130 gene sequence, the desired gp130protein is purified by a known method from the inside of the host cellor from the culture supernatant. This purified gp130 protein may be usedas a sensitizing antigen. Alternatively, a cell expressing gp130 or afusion protein of the gp130 protein and another protein may be used as asensitizing antigen.

Mammals to be immunized with a sensitizing antigen are not particularlylimited, but are preferably selected in consideration of thecompatibility with the parent cell used for cell fusion. Generally,rodents such as mice, rats, and hamsters are used.

Immunization of animals with a sensitizing antigen is performedaccording to known methods. For example, as a general method, it isperformed by injecting the sensitizing antigen intraperitoneally orsubcutaneously into mammals. Specifically, the sensitizing antigen ispreferably diluted or suspended in an appropriate amount ofphosphate-buffered saline (PBS), physiological saline or such, mixedwith an appropriate amount of a general adjuvant (e.g., Freund'scomplete adjuvant), emulsified, and then administered for several timesevery 4 to 21 days to a mammal. In addition, an appropriate carrier maybe used for the immunization with a sensitizing antigen.

Following such immunization, an increased level of the desired antibodyin serum is confirmed and then immune cells are obtained from the mammalfor cell fusion. Preferred immune cells for cell fusion include, inparticular, spleen cells.

For the mammalian myeloma cells to be used as a parent cell, i.e. apartner cell to be fused with the above immune cells, various known cellstrains, for example, P3X63Ag8.653 (Kearney, J. F. et al., J. Immunol(1979) 123, 1548-1550), P3X63Ag8U.1 (Current Topics in Microbiology andImmunology (1978) 81, 1-7), NS-1 (Kohler, G. and Milstein, C., Eur. J.Immunol. (1976) 6, 511-519), MPC-11 (Margulies, D. H. et al., Cell(1976) 8, 405-415), SP2/0 (Shulman, M. et al., Nature (1978) 276,269-270), F0 (de St. Groth, S. F. et al., J. Immunol. Methods (1980) 35,1-21), S194 (Trowbridge, I. S., J. Exp. Med. (1978) 148, 313-323), R210(Galfre, G. et al., Nature (1979) 277, 131-133), and such areappropriately used.

Basically, cell fusion of the aforementioned immune cell and myelomacell can be performed using known methods, for example, the method byMilstein et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981)73, 3-46) and such.

More specifically, the aforementioned cell fusion is achieved in generalnutrient culture medium under the presence of a cell fusion enhancingagent. For example, polyethylene glycol (PEG), Sendai virus (HVJ), andsuch are used as a fusion enhancing agent. Further, to enhance thefusion efficiency, auxiliary agents such as dimethyl sulfoxide may beadded for use according to needs.

The ratio of immune cells and myeloma cells used is preferably, forexample, 1 to 10 immune cells for each myeloma cell. The culture mediumused for the aforementioned cell fusion is, for example, the RPMI1640 orMEM culture medium, which are suitable for the proliferation of theaforementioned myeloma cells. A general culture medium used forculturing this type of cell can also be used. Furthermore, serumsupplements such as fetal calf serum (FCS) can be used in combination.

For cell fusion, the fusion cells (hybridomas) of interest are formed bymixing predetermined amounts of the aforementioned immune cell andmyeloma cell well in the aforementioned culture medium, and then addingand mixing a concentration of 30 to 60% (w/v) PEG solution (e.g., a PEGsolution with a mean molecular weight of about 1,000 to 6,000)pre-heated to about 37° C. Then, cell fusion agents and such that areunsuitable for the growth of hybridoma can be removed by repeating thesteps of successively adding an appropriate culture medium and removingthe supernatant by centrifugation.

The above hybridomas are selected by culturing cells in a generalselection culture medium, for example, HAT culture medium (a culturemedium containing hypoxanthine, aminopterin, and thymidine). Culturingin the HAT culture medium is continued for a sufficient period of time,generally for several days to several weeks, to kill cells other thanthe hybridomas of interest (unfused cells). Then, the standard limiteddilution method is performed to screen and clone hybridomas that producethe antibody of interest.

In addition to the method of immunizing a non-human animal with anantigen for obtaining the aforementioned hybridomas, a desired humanantibody that has the activity of binding to a desired antigen orantigen-expressing cell can be obtained by sensitizing a humanlymphocyte with a desired antigen protein or antigen-expressing cell invitro, and fusing the sensitized B lymphocyte with a human myeloma cell(e.g., U266) (see, Japanese Patent Application Kokoku Publication No.(JP-B) H01-59878 (examined, approved Japanese patent applicationpublished for opposition)). Furthermore, a desired human antibody can beobtained by administering the antigen or antigen-expressing cell to atransgenic animal that has a repertoire of human antibody genes and thenfollowing the aforementioned method (see, International PatentApplication Publication Nos. WO 93/12227, WO 92/03918, WO 94/02602, WO94/25585, WO 96/34096, and WO 96/33735).

The thus-prepared hybridomas which produce monoclonal antibodies can besubcultured in conventional culture medium and stored in liquid nitrogenfor a long period.

For obtaining monoclonal antibodies from the aforementioned hybridomas,the following methods may be employed: (1) method where the hybridomasare cultured according to conventional methods and the antibodies areobtained as a culture supernatant; (2) method where the hybridomas areproliferated by administering them to a compatible mammal and theantibodies are obtained as ascites; and so on. The former method ispreferred for obtaining antibodies with high purity, and the latter ispreferred for large-scale production of antibodies.

For example, the preparation of anti-IL-6 receptor antibody-producinghybridomas can be performed by the method disclosed in JP-A H03-139293.The preparation can be performed by the method of injecting a PM-1antibody-producing hybridoma into the abdominal cavity of a BALB/cmouse, obtaining ascite, and then purifying PM-1 antibody from theascite, or the method of culturing the hybridoma in an appropriatemedium (e.g., RPMI1640 medium containing 10% fetal bovine serum, and 5%BM-Condimed H1 (Boehringer Mannheim); hybridoma SFM medium (GIBCO-BRL);PFHM-II medium (GIBCO-BRL), etc.) and then obtaining PM-1 antibody fromthe culture supernatant.

A recombinant antibody can be used as a monoclonal antibody of thepresent invention, wherein the antibody is produced through geneticrecombination techniques by cloning an antibody gene from a hybridoma,inserting the gene into an appropriate vector, and then introducing thevector into a host (see, for example, Borrebaeck, C. A. K. and Larrick,J. W., THERAPEUTIC MONOCLONAL ANTIBODIES, published in the UnitedKingdom by MACMILLAN PUBLISHERS LTD., 1990).

More specifically, mRNA coding for the variable (V) region of anantibody is isolated from a cell that produces the antibody of interest,such as a hybridoma. The isolation of mRNA can be performed by preparingtotal RNA according to known methods, such as the guanidineultracentrifugation method (Chirgwin, J. M. et al., Biochemistry (1979)18, 5294-5299) and the AGPC method (Chomczynski, P. et al., Anal.Biochem. (1987) 162, 156-159), and preparing mRNA using the mRNAPurification Kit (Pharmacia) and such. Alternatively, mRNA can bedirectly prepared using the QuickPrep mRNA Purification Kit (Pharmacia).

cDNA of the antibody V region is synthesized from the obtained mRNAusing reverse transcriptase. The synthesis of cDNA may be achieved usingthe AMV Reverse Transcriptase First-strand cDNA Synthesis Kit and so on.Furthermore, to synthesize and amplify the cDNA, the 5′-RACE method(Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002;Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) using5′-Ampli FINDER RACE Kit (Clontech) and PCR may be employed. The DNAfragment of interest is purified from the obtained PCR products and thenligated with a vector DNA. Then, a recombinant vector is prepared usingthe above DNA and introduced into Escherichia coli or such, and itscolonies are selected to prepare the desired recombinant vector. Thenucleotide sequence of the DNA of interest is confirmed by, for example,the dideoxy method.

When a DNA encoding the V region of an antibody of interest is obtained,the DNA is ligated with a DNA that encodes a desired antibody constantregion (C region), and inserted into an expression vector.Alternatively, the DNA encoding the antibody V region may be insertedinto an expression vector comprising the DNA of an antibody C region.

To produce an antibody to be used in the present invention, as describedbelow, the antibody gene is inserted into an expression vector so thatit is expressed under the control of the expression regulating region,for example, enhancer and promoter. Then, the antibody can be expressedby transforming a host cell with this expression vector.

In the present invention, to decrease heteroantigenicity against humanand such, artificially modified genetic recombinant antibodies, forexample, chimeric antibodies, humanized antibodies, or human antibodies,can be used. These modified antibodies can be prepared using knownmethods.

A chimeric antibody can be obtained by ligating the antibody Vregion-encoding DNA obtained as above with a human antibody Cregion-encoding DNA, inserting the DNA into an expression vector andintroducing it into a host for production (see, European PatentApplication Publication No. EP 125023; International Patent ApplicationPublication No. WO 92/19759). This known method can be used to obtainchimeric antibodies useful for the present invention.

Humanized antibodies are also referred to as reshaped human antibodies,and are antibodies wherein the complementarity determining regions(CDRs) of an antibody from a mammal other than human (e.g., mouseantibody) are transferred into the CDRs of a human antibody. Generalmethods for this gene recombination are also known (see, European PatentApplication Publication No. EP 125023, International Patent ApplicationPublication No. WO 92/19759).

More specifically, a DNA sequence designed such that the CDRs of a mouseantibody are ligated with the framework regions (FRs) of a humanantibody is synthesized by PCR from several oligonucleotides that hadbeen produced to contain overlapping portions at their termini. Theobtained DNA is ligated with a human antibody C region-encoding DNA andthen inserted into an expression vector. The expression vector isintroduced into a host to produce the humanized antibody (see, EuropeanPatent Application Publication No. EP 239400, International PatentApplication Publication No. WO 92/19759).

The human antibody FRs to be ligated via the CDRs are selected so thatthe CDRs form a suitable antigen binding site. The amino acid(s) withinthe FRs of the antibody variable regions may be substituted as necessaryso that the CDRs of the reshaped human antibody form an appropriateantigen binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

Human antibody C regions are used for the chimeric and humanizedantibodies, and include Cγ. For example, Cγ1, Cγ2, Cγ3, or Cγ4 may beused. Furthermore, to improve the stability of the antibody or itsproduction, the human antibody C regions may be modified.

Chimeric antibodies consist of the variable region of an antibodyderived from non-human mammals and a human antibody-derived C region;and humanized antibodies consist of the CDRs of an antibody derived fromnon-human mammals and the framework regions and C regions derived from ahuman antibody. Both have reduced antigenicity in human body, and aretherefore useful as antibodies to be used in the present invention.

Preferred specific examples of humanized antibodies used in the presentinvention include a humanized PM-1 antibody (see, International PatentApplication Publication No. WO 92/19759).

Furthermore, in addition to the aforementioned method for obtaining ahuman antibody, techniques for obtaining human antibodies by panningusing a human antibody library are also known. For example, it ispossible to express the variable regions of human antibodies on thesurface of phages as single chain antibodies (scFv) by the phage displaymethod, and then select antigen-binding phages. By analyzing genes ofthe selected phages, DNA sequences coding for the human antibodyvariable regions that bind to the antigen can be determined. Once theDNA sequence of an scFv that binds to the antigen is revealed, anappropriate expression vector comprising the sequence can be constructedto obtain an human antibody. These methods are already known, and thepublications of WO 92/01047, WO 92/20791, WO93/06213, WO 93/11236, WO93/19172, WO 95/01438, and WO 95/15388 can be used as reference.

The above-constructed antibody gene can be expressed according toconventional methods. When a mammalian cell is used, the antibody genecan be expressed using a DNA in which the antibody gene to be expressedis functionally ligated to a useful commonly used promoter and a poly Asignal downstream of the antibody gene, or a vector comprising the DNA.Examples of a promoter/enhancer include the human cytomegalovirusimmediate early promoter/enhancer.

Furthermore, other promoters/enhancers that can be utilized forexpressing the antibody to be used in the present invention includeviral promoters/enhancers from retrovirus, polyoma virus, adenovirus,simian virus 40 (SV40), and such; and mammalian cell-derivedpromoters/enhancers such as human elongation factor 1α (HEF1α).

For example, when the SV40 promoter/enhancer is used, the expression canbe easily performed by following the method by Mulligan et al.(Mulligan, R. C. et al., Nature (1979) 277, 108-114). Alternatively, inthe case of the HEF1α promoter/enhancer, the method by Mizushima et al.(Mizushima, S. and Nagata S., Nucleic Acids Res. (1990) 18, 5322) can beused.

When E. coli is used, the antibody gene can be expressed by functionallyligating a conventional useful promoter, a signal sequence for antibodysecretion, and the antibody gene to be expressed. Examples of a promoterinclude the lacZ promoter, araB promoter and such. When the lacZpromoter is used, the expression can be performed according to themethod by Ward et al. (Ward, E. S. et al., Nature (1989) 341, 544-546;Ward, E. S. et al., FASEB J. (1992) 6, 2422-2427); and the araB promotermay be used according to the method by Better et al. (Better, M. et al.,Science (1988) 240, 1041-1043).

When the antibody is produced into the periplasm of E. coli, the pel Bsignal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379-4383)may be used as the signal sequence for antibody secretion. The antibodyproduced into the periplasm is isolated, and then used afterappropriately refolding the antibody structure (see, e.g., WO 96/30394).

As the replication origin, those derived from SV40, polyoma virus,adenovirus, bovine papilloma virus (BPV) and such may be used. Inaddition, for enhancing the gene copy number in a host cell system, theexpression vector may comprise the aminoglycoside phosphotransferase(APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guaninephosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (dhfr)gene, or such as a selection marker.

Any production system may be used for preparing the antibodies to beused in the present invention. The production systems for antibodypreparation include in vitro and in vivo production systems. In vitroproduction systems include those utilizing eukaryotic cells orprokaryotic cells.

Production systems using eukaryotic cells include those utilizing animalcells, plant cells, or fungal cells. Such animal cells include (1)mammalian cells, for example, CHO, COS, myeloma, baby hamster kidney(BHK), HeLa, Vero, and such; (2) amphibian cells, for example, Xenopusoocyte; and (3) insect cells, for example, sf9, sf21, Tn5, and such.Known plant cells include cells derived from Nicotiana tabacum, whichmay be cultured as callus. Known fungal cells include yeast such asSaccharomyces (e.g., S. cerevisiae), mold fungi such as Aspergillus(e.g., A. niger), and such.

Production systems using prokaryotic cells include those utilizingbacterial cells. Known bacterial cells include E. coli and Bacillussubtilis.

Antibodies can be obtained by introducing an antibody gene of interestinto these cells by transformation, and culturing the transformed cellsin vitro. The culturing is conducted according to known methods. Forexample, DMEM, MEM, RPMI1640, IMDM may be used as the culture medium,and serum supplements, such as FCS, may be used in combination.Furthermore, a cell introduced with an antibody gene may be transferredinto the abdominal cavity or such of an animal to produce an antibody invivo.

On the other hand, in vivo production systems include those utilizinganimals or plants. Production systems using animals include those thatutilize mammals or insects.

Mammals that can be used include goats, pigs, sheep, mice, bovines andsuch (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Further,insects that can be used include silkworms. When using plants, forexample, tobacco may be used.

An antibody gene is introduced into these animals or plants, and anantibody is produced in the body of the animals or plants and thenrecovered. For example, the antibody gene is prepared as a fusion geneby inserting the gene in the middle of a gene encoding a protein, suchas goat β casein, which is uniquely produced into milk. A DNA fragmentcomprising the antibody gene-inserted fusion gene is injected into agoat embryo, and the embryo is introduced into a female goat. Thedesired antibody is obtained from the milk produced from the transgenicanimal born from the goat that received the embryo, or produced fromprogenies of the animal. To increase the amount of milk that containsthe desired antibody produced from the transgenic goat, hormones may byappropriately used on the transgenic goat (Ebert, K. M. et al.,Bio/Technology (1994) 12, 699-702).

Furthermore, when a silkworm is used, it is infected with baculovirusinserted with the desired antibody gene, and the desired antibody isobtained from the body fluid of this silkworm (Maeda, S. et al., Nature(1985) 315, 592-594). Moreover, when tobacco is used, the desiredantibody gene is inserted into a plant expression vector (e.g., pMON530)and the vector is introduced into bacteria such as Agrobacteriumtumefaciens. This bacterium is used to infect tobacco (e.g., Nicotianatabacum) to obtain the desired antibody from the leaves of this tobacco(Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).

When producing an antibody in in vitro or in vivo production systems asdescribed above, DNAs encoding the antibody heavy chain (H chain) andlight chain (L chain) may be inserted into separate expression vectorsand a host is then co-transformed with the vectors. Alternatively, theDNAs may be inserted into a single expression vector for transforming ahost (see, International Patent Application Publication No. WO94/11523).

The antibodies used in the present invention may be antibody fragmentsor modified products thereof so long as they can be suitably used in thepresent invention. For example, antibody fragments include Fab, F(ab′)2,Fv, and single chain Fv (scFv) in which the Fvs of the H and L chainsare linked via an appropriate linker.

Specifically, the antibody fragments are produced by treating anantibody with an enzyme, for example, papain or pepsin, oralternatively, genes encoding these fragments are constructed,introduced into expression vectors, and expressed in an appropriate hostcell (see, e.g., Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976;Better, M. & Horwitz, A. H., Methods in Enzymology (1989) 178, 497-515;Plueckthun, A. & Skerra, A., Methods in Enzymology (1989) 178, 497-515;Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J. etal., Methods in Enzymology (1989) 121, 663-666; Bird, R. E. et al.,TIBTECH (1991) 9, 132-137).

An scFv can be obtained by linking the H-chain V region and the L-chainV region of an antibody. In the scFv, the H-chain V region and theL-chain V region are linked via a linker, preferably via a peptidelinker (Huston, J. S. et al., Proc. Natl. Acad. Sci. USA (1988) 85,5879-5883). The V regions of the H and L chains in an scFv may bederived from any of the antibodies described above. Peptide linkers forlinking the V regions include, for example, an arbitrary single chainpeptide consisting of 12 to 19 amino acid residues.

An scFv-encoding DNA can be obtained by using the DNA encoding the Hchain or its V region and the DNA encoding the L chain or its V regionof the aforementioned antibodies as templates, PCR amplifying the DNAportion that encodes the desired amino acid sequence in the templatesequence using primers that define the termini of the portion, and thenfurther amplifying the amplified DNA portion with a peptide linkerportion-encoding DNA and primer pairs that link both ends of the linkerto the H chain and L chain.

Furthermore, once an scFv-encoding DNA has been obtained, an expressionvector comprising the DNA and a host transformed with the vector can beobtained according to conventional methods. In addition, the scFv can beobtained according to conventional methods using the host.

Similarly as above, these antibody fragments can be produced from thehost by obtaining and expressing their genes. Herein, “antibody”encompasses these antibody fragments.

As a modified antibody, an antibody bound to various molecules, such aspolyethylene glycol (PEG), may also be used. Herein, “antibody”encompasses these modified antibodies. These modified antibodies can beobtained by chemically modifying the obtained antibodies. Such methodsare already established in the art.

The antibodies produced and expressed as above can be isolated from theinside or outside of the cell or from host, and purified to homogeneity.The isolation and/or purification of the antibodies used for the presentinvention can be performed by affinity chromatography. Columns to beused for the affinity chromatography include, for example, protein Acolumn and protein G column. Carriers used for the protein A columninclude, for example, HyperD, POROS, SepharoseF.F. and such. In additionto the above, other methods used for the isolation and/or purificationof common proteins may be used, and are not limited in any way.

For example, the antibodies used for the present invention may beisolated and/or purified by appropriately selecting and combiningchromatographies besides affinity chromatography, filters,ultrafiltration, salting-out, dialysis, and such. Chromatographiesinclude, for example, ion-exchange chromatography, hydrophobicchromatography, gel filtration, and such. These chromatographies can beapplied to high performance liquid chromatography (HPLC). Alternatively,reverse phase HPLC may be used.

Concentration of the antibodies as obtained above can be determined byabsorbance measurement, ELISA, or such. Specifically, the absorbance isdetermined by appropriately diluting the antibody solution with PBS(−),measuring the absorbance at 280 nm, and calculating the concentration(1.35 OD=1 mg/ml). Alternatively, when using ELISA, the measurement canbe performed as follows. Specifically, 100 μl of goat anti-human IgG(TAG) diluted to 1 μg/ml with 0.1 M bicarbonate buffer (pH 9.6) is addedto a 96-well plate (Nunc) and incubated overnight at 4° C. to immobilizethe antibody. After blocking, 100 μl of an appropriately dilutedantibody of the present invention or an appropriately diluted samplecomprising the antibody, and human IgG (CAPPEL) are added as a standard,and incubated for one hour at room temperature.

After washing, 100 μl of 5,000× diluted alkaline phosphatase-labeledanti-human IgG (BIO SOURCE) is added and incubated for one hour at roomtemperature. After another wash, substrate solution is added andincubated, and the absorbance at 405 nm is measured using MICROPLATEREADER Model 3550 (Bio-Rad) to calculate the concentration of theantibody of interest.

IL-6 variants used in the present invention are substances that have theactivity to bind to an IL-6 receptor and which do not transmit IL-6biological activity. That is, the IL-6 variants compete with IL-6 tobind to IL-6 receptors, but fail to transmit IL-6 biological activity,hence blocking IL-6-mediated signal transduction.

The IL-6 variants are produced by introducing mutation(s) throughsubstitution of amino acid residues in the amino acid sequence of IL-6.The origin of IL-6 used as the base of the IL-6 variants is not limited;however, it is preferably human IL-6 when considering its antigenicityand such.

More specifically, amino acid substitution is performed by predictingthe secondary structure of the IL-6 amino acid sequence using knownmolecular modeling programs (e.g., WHATIF; Vriend et al., J. Mol.Graphics (1990) 8, 52-56), and further assessing the influence of thesubstituted amino acid residue(s) on the whole molecule. Afterdetermining the appropriate amino acid residue to be substituted,commonly performed PCR methods are carried out using the human IL-6gene-encoding nucleotide sequence as a template to introduce mutationsso that amino acids are substituted, and thereby an IL-6variant-encoding gene is obtained. If needed, this gene is inserted intoan appropriate expression vector, and the IL-6 variant can be obtainedby applying the aforementioned methods for expression, production, andpurification of recombinant antibodies.

Specific examples of the IL-6 variants are disclosed in Brakenhoff etal., J. Biol. Chem. (1994) 269, 86-93, Savino et al., EMBO J. (1994) 13,1357-1367, WO 96/18648, and WO 96/17869.

Partial peptides of IL-6 and partial peptides of IL-6 receptors to beused in the present invention are substances that have the activity tobind to IL-6 receptors and IL-6, respectively, and which do not transmitIL-6 biological activity. Namely, by binding to and capturing an IL-6receptor or IL-6, the IL-6 partial peptide or the IL-6 receptor partialpeptide specifically inhibits IL-6 from binding to the IL-6 receptor. Asa result, the biological activity of IL-6 is not transmitted, andtherefore IL-6-mediated signal transduction is blocked.

The partial peptides of IL-6 or IL-6 receptor are peptides that comprisepart or all of the amino acid sequence of the region of the IL-6 or IL-6receptor amino acid sequence that is involved in the binding of IL-6 andIL-6 receptor. Such peptides usually comprise 10 to 80, preferably 20 to50, more preferably 20 to 40 amino acid residues.

The IL-6 partial peptides or IL-6 receptor partial peptides can beproduced according to generally known methods, for example, geneticengineering techniques or peptide synthesis method, by specifying theregion of the IL-6 or IL-6 receptor amino acid sequence that is involvedin the binding of IL-6 and IL-6 receptor, and using a portion or wholeof the amino acid sequence of the specified region.

When preparing an IL-6 partial peptide or IL-6 receptor partial peptideby a genetic engineering method, a DNA sequence encoding the desiredpeptide is inserted into an expression vector, and then the peptide canbe obtained by applying the aforementioned methods for expressing,producing, and purifying recombinant antibodies.

To produce an IL-6 partial peptide or IL-6 receptor partial peptide bypeptide synthesis methods, the generally used peptide synthesis methods,for example, solid phase synthesis methods or liquid phase synthesismethods may be used.

Specifically, the synthesis can be performed following the methoddescribed in “Continuation of Development of Pharmaceuticals, Vol. 14,Peptide Synthesis (in Japanese) (ed. Haruaki Yajima, 1991, HirokawaShoten)”. As a solid phase synthesis method, for example, the followingmethod can be employed: the amino acid corresponding to the C terminusof the peptide to be synthesized is bound to a support that is insolublein organic solvents, then elongating the peptide strand by alternatelyrepeating (1) the reaction of condensing amino acids whose α-aminogroups and branch chain functional groups are protected with appropriateprotecting groups one at a time in a C to N-terminal direction; and (2)the reaction of removing protecting groups from the α-amino groups ofthe resin-bound amino acid or peptide. The solid phase peptide synthesisis broadly classified into the Boc method and the Fmoc method based onthe type of protecting group used.

After the protein of interest is synthesized as above, deprotectionreaction and reaction to cleave the peptide strand from the support arecarried out. For the cleavage reaction of the peptide strand, ingeneral, hydrogen fluoride or trifluoromethane sulfonic acid is used forthe Boc method, and TFA for the Fmoc method. According to the Bocmethod, for example, the above-mentioned protected peptide resin istreated in hydrogen fluoride under the presence of anisole. Then, thepeptide is recovered by removing the protecting group and cleaving thepeptide from the support. By freeze-drying the recovered peptide, acrude peptide can be obtained. On the other hand, in the Fmoc method,for example, the deprotection reaction and the reaction to cleave thepeptide strand from the support can be performed in TFA by a similarmethod as described above.

The obtained crude peptide can be separated and/or purified by applyingHPLC. Elution may be performed under optimum conditions using awater-acetonitrile solvent system, which is generally used for proteinpurification. The fractions corresponding to the peaks of the obtainedchromatographic profile are collected and freeze-dried. Thus, purifiedpeptide fractions are identified by molecular weight analysis via massspectrum analysis, amino acid composition analysis, amino acid sequenceanalysis, or such.

Specific examples of IL-6 partial peptides and IL-6 receptor partialpeptides are disclosed in JP-A H02-188600, JP-A H07-324097, JP-AH08-311098, and U.S. Pat. No. 5,210,075.

The antibodies used in the present invention may also be conjugatedantibodies which are bound to various molecules, such as polyethyleneglycol (PEG), radioactive substances, and toxins. Such conjugatedantibodies can be obtained by chemically modifying the obtainedantibodies. Methods for modifying antibodies are already established inthe art. The “antibodies” of the present invention encompass theseconjugated antibodies.

The IL-6 inhibitors of the present invention can be used to promotemuscle regeneration. Herein, “muscle regeneration” refers to recovery ofdamaged or atrophied muscles to their original condition. The recoveryof muscles to their original condition means that the volume or numberof muscle fibers, or the property of muscle tissues (tensiondevelopment, endurance capacity, metabolic properties, elasticity,and/or flexibility) returns to the level before the damage or atrophy.Herein, preferred examples of “muscle atrophy” include muscle atrophyoccurring in the absence of gravitational loading, muscle atrophy causedby disuse of muscles, muscle atrophy accompanying chronic inflammatorydiseases such as rheumatoid arthritis, muscle atrophy in congenitalmuscular diseases, and others.

In the present invention, the “muscle or muscle tissue” is notparticularly limited, and it may be any one of skeletal muscle cell,cardiac muscle cell, smooth muscle cell, and/or myoepithelial cell.

The process of muscle regeneration, in which satellite cells involve, isdescribed below. It is thought that satellite cells in adult muscletissues generally have their cell division arrested or aredifferentiating slowly. They become activated and start to proliferatewhen muscles such as skeletal muscles are damaged. Satellite cells thathave proliferated and passed through the basement membrane differentiateinto precursor cells called myoblasts and migrate to the damaged siteswhile actively proliferating and differentiating. The myoblasts arrangethemselves on the basement membrane around the damaged muscle fibers,then invade into the inner side of the basal membrane, and fuse witheach other or with remnant muscle fibers to form myotubes. The myotubesachieve structural maturation and become adult muscle tissue.

In the present invention, “muscle regeneration” may refer to theformation of adult muscle tissue through the process described above.

In the present invention, “promotion of muscle regeneration” means thatthe progression of the muscle regeneration, described above, isaccelerated. Moreover, promotion of the activation of satellite cells,involved in muscle regeneration, can also be assumed to be equivalent topromotion of muscle regeneration. The phrase “promoting the activationof satellite cells” means promoting the recruitment, proliferation, ordifferentiation of satellite cells.

In the present invention, whether muscle regeneration has been promotedcan be confirmed by measuring the volume and/or number of muscle fibers.When the volume or number of muscle fibers is increased by administeringthe agents of the present invention, muscle regeneration can beconsidered to be promoted. The volume or number of muscle fibers can bemeasured by known methods or by the methods described in Examples.

In addition, it can also be confirmed, whether muscle regeneration ispromoted, by measuring the number of mitotic active satellite cells (thepercentage of mitotic active satellite cells/total satellite cells).When the number of mitotic active satellite cells is increased byadministration of the agents in the present invention, it can beconsidered that muscle regeneration is promoted. The number of mitoticactive satellite cells can be measured by known methods or also by themethods described in Examples.

In the present invention, the activity of IL-6 inhibitors in inhibitingthe transduction of IL-6 signal can be evaluated by conventionalmethods. Specifically, IL-6 is added to cultures of IL-6-dependent humanmyeloma cell lines (S6B45 and KPMM2), human Lennert T lymphoma cell lineKT3, or IL-6-dependent cell line MH60.BSF2; and the ³H-thymidine uptakeby the IL-6-dependent cells is measured in the presence of an IL-6inhibitor. Alternatively, IL-6 receptor-expressing U266 cells arecultured, and ¹²⁵I-labeled IL-6 and an IL-6 inhibitor are added to theculture at the same time; and then ¹²⁵I-labeled IL-6 bound to the IL-6receptor-expressing cells is quantified. In addition to the IL-6inhibitor group, a negative control group that does not contain the IL-6inhibitor is included in the assay system described above. The activityof the IL-6 inhibitor to inhibit IL-6 can be evaluated by comparing theresults of both groups.

As shown below in the Examples, administration of an anti-IL-6 receptorantibody was found to promote muscle regeneration. This finding suggeststhat IL-6 inhibitors, such as anti-IL-6 receptor antibodies, are usefulas the agents for facilitating muscle regeneration.

Subjects to be administered with the agents of the present invention forfacilitating muscle regeneration are mammals. The mammals are preferablyhumans.

The agents of the present invention for facilitating muscle regenerationcan be administered as pharmaceuticals, and may be administeredsystemically or locally via oral or parenteral administration. Forexample, intravenous injection such as drip infusion, intramuscularinjection, intraperitoneal injection, subcutaneous injection,suppository, enema, oral enteric tablets, or the like can be selected.An appropriate administration method can be selected depending on thepatient's age and symptoms. The effective dose per administration isselected from the range of 0.01 to 100 mg/kg body weight. Alternatively,the dose may be selected from the range of 1 to 1000 mg/patient,preferably from the range of 5 to 50 mg/patient. A preferred dose andadministration method are as follows: for example, when an anti-IL-6receptor antibody is used, the effective dose is an amount such thatfree antibody is present in the blood. Specifically, a dose of 0.5 to 40mg/kg body weight/month (four weeks), preferably 1 to 20 mg/kg bodyweight/month is administered via intravenous injection such as dripinfusion, subcutaneous injection or such, once to several times a month,for example, twice a week, once a week, once every two weeks, or onceevery four weeks. The administration schedule may be adjusted by, forexample, extending the administration interval of twice a week or once aweek to once every two weeks, once every three weeks, or once every fourweeks, while monitoring the condition after administration and changesin the blood test values.

In the present invention, the agents for facilitating muscleregeneration may contain pharmaceutically acceptable carriers, such aspreservatives and stabilizers. The “pharmaceutically acceptablecarriers” refer to materials that can be co-administered with anabove-described agent; and may or may not itself produce theabove-described effect of facilitating muscle regeneration.Alternatively, the carriers may be materials that do not have the effectof facilitating muscle regeneration, but produce an additive orsynergistic effect when used in combination with an IL-6 inhibitor.

Such pharmaceutically acceptable materials include, for example, sterilewater, physiological saline, stabilizers, excipients, buffers,preservatives, detergents, chelating agents (EDTA and such), andbinders.

In the present invention, detergents include non-ionic detergents, andtypical examples of such include sorbitan fatty acid esters such assorbitan monocaprylate, sorbitan monolaurate, and sorbitanmonopalmitate; glycerin fatty acid esters such as glycerinmonocaprylate, glycerin monomyristate and glycerin monostearate;polyglycerin fatty acid esters such as decaglyceryl monostearate,decaglyceryl distearate, and decaglyceryl monolinoleate; polyoxyethylenesorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate,polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylenesorbitan trioleate, and polyoxyethylene sorbitan tristearate;polyoxyethylene sorbit fatty acid esters such as polyoxyethylene sorbittetrastearate and polyoxyethylene sorbit tetraoleate; polyoxyethyleneglycerin fatty acid esters such as polyoxyethylene glycerylmonostearate; polyethylene glycol fatty acid esters such as polyethyleneglycol distearate; polyoxyethylene alkyl ethers such as polyoxyethylenelauryl ether; polyoxyethylene polyoxypropylene alkyl ethers such aspolyoxyethylene polyoxypropylene glycol, polyoxyethylenepolyoxypropylene propyl ether, and polyoxyethylene polyoxypropylenecetyl ether; polyoxyethylene alkyl phenyl ethers such as polyoxyethylenenonylphenyl ether; polyoxyethylene hardened castor oils such aspolyoxyethylene castor oil and polyoxyethylene hardened castor oil(polyoxyethylene hydrogenated castor oil); polyoxyethylene beeswaxderivatives such as polyoxyethylene sorbit beeswax; polyoxyethylenelanolin derivatives such as polyoxyethylene lanolin; and polyoxyethylenefatty acid amides and such with an HLB of 6 to 18, such aspolyoxyethylene stearic acid amide.

Detergents also include anionic detergents, and typical examples of suchinclude, for example, alkylsulfates having an alkyl group with 10 to 18carbon atoms, such as sodium cetylsulfate, sodium laurylsulfate, andsodium oleylsulfate; polyoxyethylene alkyl ether sulfates in which thealkyl group has 10 to 18 carbon atoms and the average molar number ofadded ethylene oxide is 2 to 4, such as sodium polyoxyethylene laurylsulfate; alkyl sulfosuccinate ester salts having an alkyl group with 8to 18 carbon atoms, such as sodium lauryl sulfosuccinate ester; naturaldetergents, for example, lecithin; glycerophospholipids;sphingo-phospholipids such as sphingomyelin; and sucrose fatty acidesters in which the fatty acids have 12 to 18 carbon atoms.

One, two or more of the detergents described above can be combined andadded to the agents of the present invention. Detergents that arepreferably used in the preparations of the present invention includepolyoxyethylene sorbitan fatty acid esters, such as polysorbates 20, 40,60, and 80. Polysorbates 20 and 80 are particularly preferred.Polyoxyethylene polyoxypropylene glycols, such as poloxamer (PluronicF-68® and such), are also preferred.

The amount of detergent added varies depending on the type of detergentused. When polysorbate 20 or 80 is used, the amount is in general in therange of 0.001 to 100 mg/ml, preferably in the range of 0.003 to 50mg/ml, more preferably in the range of 0.005 to 2 mg/ml.

In the present invention, buffers includes phosphate, citrate buffer,acetic acid, malic acid, tartaric acid, succinic acid, lactic acid,potassium phosphate, gluconic acid, capric acid, deoxycholic acid,salicylic acid, triethanolamine, fumaric acid, and other organic acids;and carbonic acid buffer, Tris buffer, histidine buffer, and imidazolebuffer.

Liquid preparations may be formulated by dissolving the agents inaqueous buffers known in the field of liquid preparations. The bufferconcentration is in general in the range of 1 to 500 mM, preferably inthe range of 5 to 100 mM, more preferably in the range of 10 to 20 mM.

The agents of the present invention may also comprise otherlow-molecular-weight polypeptides; proteins such as serum albumin,gelatin, and immunoglobulin; amino acids; sugars and carbohydrates suchas polysaccharides and monosaccharides, sugar alcohols, and such.

Herein, amino acids include basic amino acids, for example, arginine,lysine, histidine, and ornithine, and inorganic salts of these aminoacids (preferably hydrochloride salts, and phosphate salts, namelyphosphate amino acids). When free amino acids are used, the pH isadjusted to a preferred value by adding appropriate physiologicallyacceptable buffering substances, for example, inorganic acids, inparticular hydrochloric acid, phosphoric acid, sulfuric acid, aceticacid, and formic acid, and salts thereof. In this case, the use ofphosphate is particularly beneficial because it gives quite stablefreeze-dried products. Phosphate is particularly advantageous whenpreparations do not substantially contain organic acids, such as malicacid, tartaric acid, citric acid, succinic acid, and fumaric acid, or donot contain corresponding anions (malate ion, tartrate ion, citrate ion,succinate ion, fumarate ion, and such). Preferred amino acids arearginine, lysine, histidine, and ornithine. Furthermore, it is possibleto use acidic amino acids, for example, glutamic acid and aspartic acid,and salts thereof (preferably sodium salts); neutral amino acids, forexample, isoleucine, leucine, glycine, serine, threonine, valine,methionine, cysteine, and alanine; and aromatic amino acids, forexample, phenylalanine, tyrosine, tryptophan, and its derivative,N-acetyl tryptophan.

Herein, sugars and carbohydrates such as polysaccharides andmonosaccharides include, for example, dextran, glucose, fructose,lactose, xylose, mannose, maltose, sucrose, trehalose, and raffinose.

Herein, sugar alcohols include, for example, mannitol, sorbitol, andinositol.

When the agents of the present invention are prepared as aqueoussolutions for injection, the agents may be mixed with, for example,physiological saline, and/or isotonic solution containing glucose orother auxiliary agents (such as D-sorbitol, D-mannose, D-mannitol, andsodium chloride). The aqueous solutions may be used in combination withappropriate solubilizing agents (such as alcohols (ethanol and such),polyalcohols (propylene glycol, PEG, and such), or non-ionic detergents(polysorbate 80 and HCO-50)).

The agents may further comprise, if required, diluents, solubilizers, pHadjusters, soothing agents, sulfur-containing reducing agents,antioxidants, and such.

Herein, the sulfur-containing reducing agents include, for example,compounds comprising sulfhydryl groups, such as N-acetylcysteine,N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine,thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodiumthiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbonatoms.

Moreover, the antioxidants in the present invention include, forexample, erythorbic acid, dibutylhydroxy toluene, butylhydroxy anisole,α-tocopherol, to copherol acetate, L-ascorbic acid and salts thereof,L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium hydrogensulfite, sodium sulfite, triamyl gallate, propyl gallate, and chelatingagents such as disodium ethylenediamine tetraacetate (EDTA), sodiumpyrophosphate, and sodium metaphosphate.

If required, the agents may be encapsulated in microcapsules(microcapsules of hydroxymethylcellulose, gelatin,poly[methylmethacrylic acid] or such) or prepared as colloidal drugdelivery systems (liposome, albumin microspheres, microemulsion,nano-particles, nano-capsules, and such) (see “Remington'sPharmaceutical Science 16^(th) edition”, Oslo Ed., 1980, and the like).Furthermore, methods for preparing agents as sustained-release agentsare also known, and are applicable to the present invention (Langer etal., J. Biomed. Mater. Res. 1981, 15: 167-277; Langer, Chem. Tech. 1982,12: 98-105; U.S. Pat. No. 3,773,919; European Patent Application No.(EP) 58,481; Sidman et al., Biopolymers 1983, 22: 547-556; and EP133,988).

Pharmaceutically acceptable carriers used are appropriately selectedfrom those described above or combined depending on the type of dosageform, but are not limited thereto.

The present invention relates to methods for promoting muscleregeneration in subjects, which comprise the step of administering anIL-6 inhibitor to the subjects.

Herein, the “subject” includes organisms with an atrophied muscle,organisms with a damaged muscle, and body parts of these organisms. Theorganisms are not particularly limited and include animals (for example,humans, domestic animals, and wild animals). The “body parts of anorganism” are not particularly limited; however, they preferably includemuscle tissues, more preferably skeletal muscles and sites surroundingthe skeletal muscles.

Herein, “administering” includes oral and parenteral administrations.Oral administration includes administering in the form of an oralpreparation. Oral preparations can be selected from dosage forms such asgranules, powder, tablets, capsules, solutions, emulsions, orsuspensions.

Parenteral administrations include administration in an injectable form.Injections include intravenous injections such as drip infusions,subcutaneus injections, muscle injections, and intraperitonealinjections. Moreover, the effects of the methods of the presentinvention can be achieved by introducing, into the body, genescomprising the oligonucleotides to be administered using gene therapymethods. The agents of the present invention can also be administeredlocally to the regions for which treatment is desired. They can also beadministered by, for example, local injection during surgery, usingcatheters, or target gene delivery of DNAs encoding the inhibitors ofthe present invention. The agents of the present invention may beadministered simultaneously, or at a different time point, with knowntherapeutic methods for muscle regeneration.

All prior art references cited herein are incorporated by reference intothis description.

EXAMPLES

Hereinbelow, the present invention will be specifically described withreference to the Examples, but it is not to be construed as beinglimited thereto.

Example 1

Decreased regulation of the immune mechanism during exposure to thespace environment is a serious problem for astronauts. C2C12 cells werecultured in a differentiation medium containing MR16-1 (an anti-mouseIL-6 receptor monoclonal antibody) at a concentration of 15 ng/ml, 150ng/ml, 1.5 μg/ml, 15 μg/ml, or 150 μg/ml in phosphate-buffered saline(PBS) to assess the effect of inhibiting the IL-6 signaling pathway onmuscle cell growth. Control cells were cultured in a medium withoutMR16-1.

After 3 days of culture, half of the cells were fixed with 10% formalinand proteins involved in muscle regeneration (MyoD, myogenin, myogenicregulatory factor proteins, and myosin heavy chain) were detectedimmunohistochemically. The remaining cells were lysed in lysis buffercontaining 1% Triton, and expressions of M-cadherin, phospho-p38, andMyoD, which are muscle differentiation markers, were confirmed byWestern blot analyses.

As a result, the proliferation of C2C12 cells was suppressed by theaddition of MR16-1. Meanwhile, treatment of cells with MR16-1 at aconcentration of 150 ng/ml or higher increased the percentagedistribution of C2C12 cells expressing MyoD, myogenin, myogenicregulatory factor proteins, and myosin heavy chain as compared withPBS-treated cells. Further, the expression levels of M-cadherin,phospho-p38, and MyoD, which are muscle differentiation markers,increased in MR16-1 treated cells (FIG. 1). These results revealed thatthe immune system plays an important role in the development and/orgrowth of muscle fibers through the IL-6 signaling pathway.

Example 2

Next, changes in the properties of satellite cells in whole singlefibers of soleus muscle, sampled from tendon to tendon, following MR16-1treatment with or without gravitational loading were investigated inmale mice (C57BL/6J Jcl).

MR16-1 or PBS was intraperitoneally (i.p.) injected into mice at aconcentration of 2 mg/mouse before seven days of hind-limb suspension orseven days of reloading. The collected muscles were dipped in cellbanker(Nihon Zenyaku) and frozen at −80° C., and then thawed at 35° C. Then,single muscle fibers were collected following collagenase digestion inDulbecco's Modified Eagle's Medium supplemented with 20 μM5′-bromo-2′-deoxyuridine (BrdU), 0.2% type I collagenase, 1%antibiotics, and 10% new-born calf serum (35° C.) for 4 hours. Themuscle fibers were incubated with an M-cadherin- or BrdU-specificantibody, and stained with fluorescein or rhodamine, respectively.M-cadherin-positive (quiescent, resting stage) or BrdU-positive (mitoticactive stage) satellite cells were analyzed using FV-300 confocal lasermicroscope (Olympus).

As a result, MR16-1 treatment produced no specific effect on musclefiber atrophy or decrease in satellite cell number associated with theabsence of a load (FIG. 2). However, the number of mitotic activesatellite cells in response to reloading was increased by MR16-1treatment (FIG. 2).

Since satellite cells play an important role in the plasticity of musclefibers, IL-6 inhibition was suggested to be a potential method forpromoting muscle regeneration.

Example 3

Satellite cells grown in MR16-1-administered culture medium are labeledwith green fluorescent protein (GFP), and the cells are injected intomuscle tissues or veins of animals with damaged or atrophied muscles.The effects of administering the cells to animals on the recovery orregeneration of their muscle tissues are assessed by biochemical and/orimmunohistochemical analyses.

INDUSTRIAL APPLICABILITY

The present inventors discovered that specific inhibition of IL-6, usingan IL-6 receptor antibody, can promote regeneration of muscles that havemuscle atrophy caused by absence of gravitational loading or disusemuscle atrophy. Therefore, the agents of the present invention forpromoting muscle regeneration are considered to be applicable as themethods for preventing or promoting recovery from the muscle atrophycaused by bedrest, plaster cast immobilization, or by space travel. Theagents are also considered to be applicable for the promotion ofregeneration of muscles after damage, atrophy accompanying chronicinflammatory diseases such as rheumatoid arthritis, and/or congenitalmuscular diseases.

There has been no therapeutic agent that promotes muscle regeneration.However, with the findings of the present invention, it is thoughtpossible to promote muscle regeneration using agents.

The invention claimed is:
 1. A method for promoting muscle regenerationin a subject affected with muscle atrophy, which comprises the steps of:selecting a subject having muscle atrophy caused by an absence ofgravitational loading or disuse muscle atrophy; subsequentlyadministering to the subject an effective amount of an IL-6 inhibitorantibody that binds to an IL-6 receptor, inhibits IL-6 from binding tothe IL-6 receptor, and further inhibits the IL-6 signaling pathway; andsubsequently re-loading the subject's atrophied muscles for at least 7days.
 2. The method of claim 1, wherein the antibody is a monoclonalantibody.
 3. The method of claim 1, wherein the antibody is an antibodythat recognizes human IL-6 receptor.
 4. The method of claim 1, whereinthe antibody is a recombinant antibody.
 5. The method of claim 1,wherein the antibody is a chimeric, humanized, or human antibody.
 6. Themethod of claim 1, wherein the antibody is MR16-1, PM-1, AUK12-20,AUK64-7, or AUK146-15.
 7. The method of claim 1, wherein the selectingstep further comprises selecting a subject having muscle atrophy causedby a microgravity space environment, long-term bed rest, or a plastercast-immobilized state.